Quantitative analyte assay device and method

ABSTRACT

The present invention related to a quantitative assay device and a method for the determination of an analyte, based on a test strip, which contains a porous test membrane allowing for capillary flow of the analyte and complexes of the analyte, a porous upstream membrane in fluid connection with the test membrane and a porous downstream membrane in fluid connection with the test membrane, wherein the test membrane contains two bands having deposited on there high and low concentrations of different calibrator agents and a test band capable of reacting with conjugated analyte complexes giving rise to a measurable signal.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation under 35 U.S.C. §120 of U.S. patentapplication Ser. No. 13/199,894, filed Sep. 12, 2011 , now U.S. Pat. No.8,900,881, issued Dec. 2, 2014, which is a continuation-in-part of U.S.patent application Ser. No. 12/317,814, filed Dec. 30, 2008, now U.S.Pat. No. 8,455,263, issued Jun. 4, 2013, the entire contents each ofwhich are incorporated herein by reference.

FIELD OF INVENTION

The present invention relates to methods and devices for assayingbiological fluid samples and particularly bodily fluid samples. Moreparticularly the invention relates to methods and devices for detectingand quantitatively measuring the concentration of an analyte in asample.

BACKGROUND

Quantitative analysis of fluid samples including antigens, antibodieshormones, and other secreted proteins as well as other types of proteinsfrequently provides critical diagnostic data. The concept ofimmunoassays is well known in the art and is based on reagentimpregnated capillary membranes in which an immobilized reagent in thedetection zone of the strip binds to analytes conjugated with visuallabel such as a latex particle or metal containing compounds. Inaddition U.S. Pat. Nos. 3,654,090, 3,791,392 and 3,817,838 discloseenzyme labels and the means to detect such, and U.S. Pat. No. 4,062,733discloses radioactive labels. Preferred labels are derived from metalsols. Labels such as prepared from a gold sol are bound to a ligandcapable of further binding or conjugating to an analyte or other ligandor antibody. Test strips have been used in qualitative assays in a largenumber of applications involving analytes contained in such bodilyfluids as urine and blood. Probably the earliest and most common use ofassays is in detecting human chorionic gonatropin as an indicator ofpregnancy in humans. These assay devices are based on test strips wherethe assay is performed and results read in a single step, e.g. where theliquid sample is placed onto a porous, membrane, any analyte of interesttherein binds a corresponding ligand, and the results are indicatedvisually in a detection zone separate from the sample loading zonethrough formation of a specific complex. The test strips are normallycontained in a dipstick or cassette allowing the user to conduct thetest without any additional equipment. Such devices are disclosed inU.S. Pat. Nos. 4,231,601, 4235,601 and 5,384,264 . Assay devices thatcan be used with whole blood are disclosed in U.S. Pat. No. 5,821,073.

Known assay devices generally involve a sandwich assay or a competitiveassay. In a sandwich assay, analyte of interest present in a fluidsample interacts with a ligand bound label or tracer resulting in acomplex of analyte and label. This can occur prior to adding the analyteto the test strip or the label bound ligand can be removably depositedon the porous membrane forming the test strip. The test strip allows thelabel-bound analyte to move by capillary action to a sample capture zonewhere the analyte complex is retained by virtue of being bound to theimmobilized capture ligand normally an antibody to the analyte. In thecase of a gold label the presence of the analyte is indicated by colorformation at the analyte test zone. Preferably the assay device alsocontains a second immobilized ligand band, generally identified as acontrol band capable of binding to label ligand even in the absence ofan analyte, to show that the strip is functioning properly.

In a competitive or inhibition assay both the analyte and the labelligand are capable of reacting with the immobilized ligand in the samplecapture zone and thus, both the analyte and the label bound ligandcompete for attachment to the immobilized ligand in the sample capturezone. Any analyte present would displace the binding of a labeledligand. The presence of a signal in the sample capture zone would thusindicate a negative result.

Most of the assay devices based on test strips determine the presence orabsence of an analyte but do not provide but a very rough measure of thequantity of an analyte present in fluid sample. Even those that give aquantitative measurement by incorporating a standard in the test devicesuffer from inaccuracies resulting from temperature, air moisture, flowtime variations, strip to strip variations and signal strengthvariations. These and other deficiencies of the prior art assay devicesare overcome by the chromatographic assay device of the presentinvention, which provides a rapid effective, and efficient quantitativeanalysis of fluid samples.

SUMMARY OF THE INVENTION

The present invention provides an assay device for and a method ofmeasuring the amount of an analyte in a fluid sample using a solid phasechromatographic assay, such as a sandwich assay, in Which one or moreanalyte of interest is bound to a label conjugate and is also bound to acapture reagent in a test band as part of a specific binding pair. Inaddition to the test band, the microporous test strip membranes of thepresent invention contain at least two different standard bands ofimmobilized calibrator agents capable of binding to their own pair labelreagents. The labeled reagents captured at the standard bands by thecalibrator agents are used to create a template against which to measurethe concentration of an analyte in a sample bound in the test band. Theimmobilized calibrator agents in the standard bands are contacted withthe corresponding label reagents as the fluid sample passes through theassay strip. Any analyte of interest present in the fluid sample isbound at the sample capture band. Based on the relative intensity of thelabel bound in these bands an accurate concentration of analyte can beobtained. Thus the density or intensity of a label such as a gold solconjugate in the various sample capture bands on the membrane stripreflects the amount of the analyte present as do the standard bandswhich then reflect the density of the calibrator agent bound and whichhas a known concentration. It is critical to an determination of theexact concentration of the analyte in the sample that the correct amountof calibrator agent be immobilized within a standard deviation of lessthan 0.05%. Commercially available optical readers can then convert thereflected light intensity of the sample into concentration as measuredon the basis of the curve derived from the reflected intensity of thestandard bands of known concentrations. A competitive assay, in whichthe analytes of interest is competitive with a capture reagent in thetest band to a label conjugate as part of a specific binding pair. Theassay device of the present invention can be used to measure theconcentration of analytes contained in any liquid sample includingblood, urine, water and oils.

The present invention, comprises a lateral flow assay method and systemincluding a test strip and/or a cassette for holding the test strip, fordetermination of the presence and/or quantity of analytes in samples,including but not limited to biological or other samples containingmaterials including antigens, antibodies, hormones and other secretedproteins, cell surface proteins, transmembrane proteins, glycoproteins,enzymes, proteins associated with cells and other proteins, proteinsassociated with pathogens such as bacteria, viruses, and fungi,carbohydrates, drugs, peptides, toxins, nucleic acids, small molecules,and aptamers. This novel assay or system can detect and/or quantitateanalytes in small volumes of samples. Generally, the sample volume isless than about 100 μl. Most preferably, the sample volume is about 40μl. This assay or system can also separate cells from fluid in a sample,such as red blood cells or white blood cells or other cell types. Theassay or system also provides low background noise and is highlyefficient.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is an exploded view of the test strip of the invention.

FIG. 2 is a perspective view of a test strip of the invention.

FIG. 3 is a perspective view of a cassette device of the invention.

FIG. 4 is an exploded view of a cassette device of the invention.

DETAILED DESCRIPTION OF INVENTION

A test strip constructed in accord with the invention for the use withwhole blood samples is shown in FIGS. 1-4. A red or white blood celllayered mesh trap 1 is shown as overlying a sample introduction membrane6 and 13 that also contains downstream from the sample addition point 7the label ligands required to bind at the test zone 4 and at thecalibrator or standard bands 3 and 5 on the microporous test membrane 2.Access to cell trap 1 for application of test sample is obtained throughopening 7 in cover 8 in FIGS. 3 and 4. The test strip 2 also includes aterminal sample flow zone 10 distal to the test and standard zones. Eachof the above mention zones are in fluid communication-with each other.All of the components described with respect to the test strip ofinvention may be encased in a fluid tight housing composed of a solidplastic cover 8 with view window 14 which fits tightly over solidplastic base 12 as shown in FIG. 3. Opening 9 in the cassette coverallows for the introduction of a buffer or other solution aiding in thecapillary flow of the sample.

For ease of understanding, the following definitions will applythroughout this description:

-   1 . The term “antigen” as used herein refers to any analyte, which    is capable of binding antibodies. Antigens may comprise, without    limitation, chemical compounds, polypeptides, carbohydrates, nucleic    acids, lipids, and the like, including viral particles, viral    subunits, bacterial and parasite surface antigens, and host proteins    that may be diagnostic of the subject's condition.-   2 . A “binder” refers to a ligand for the analyte as in the format    of a sandwich assay, or a ligand for both the analyte and the tracer    or label as in the format of a competitive assay. A binder can be    chosen from a group of molecules or compounds capable of binding the    analyte, such as an antigen to the antibody analyte, or an antibody    to the antigen analyte.-   3 . A “test zone or band” refers to an area in which a binder or the    analyte is attached, movably or immovably, such as to the analyte    test strip portion of an assay device.-   4 . The term “test strip” or “assay strip” refers to a porous    membrane capable of capillary flow of the fluid sample containing    the bound analytes and other antigens involved in the determination    of the analyte concentration. Common porous membranes are    fiberglass, porous nitrocellulose or polyethylene. An “analyte test    strip” of the invention consists of, collectively, all of the zone    supporting membranes and any filters of the assay device.-   5 . A “tracer” refers to a ligand for the analyte or the binder    labeled with a detectable label, preferably a visually readable    particulate label, such as colloidal gold, latex and liposomes    including dye, carbon black, and the like.-   6 . A “sample loading or application zone” refers to an area of an    analyte test strip on which a fluid analyte sample is applied for    migration to the test bands.-   7 . A “fluid analyte sample” can be any fluid suspected of    containing analyte of interest for which a particular assay is    specific. Test sample may represent any body fluid, including urine,    blood, sweat, lymph, intraperitoneal fluid, crude tissue extract or    homogenate, derived from a fetus, neonate, juvenile or adult    subject; a non-biological fluid such as water from some ecological    niche, e.g., a river or a lake; or a solution used in a laboratory.-   8 . A “label” is a molecule or compound, which directly or    indirectly mediates the formation of a signal (such as a color    change), which is used in assay to indicate the presence, absence or    concentration range of analyte of interest in a test sample. Labels    may include enzymes, fluorescers, liposomes, erythrocyte ghosts,    polymer microcapsules, color polymer particles (latex), and    preferably includes sols of metal-containing compounds. A wide    variety of patents and patent applications provide an extensive    literature of different techniques for producing detectible signals    in immunoassays. The following list of United States patents is    merely illustrative of the type of label which can find application    in this invention: U.S. Pat. No. 3,646,346 discloses radioactive    labels; U.S. Pat. Nos. 3,654,090, 3,791,932, and 3,817,838 disclose    enzyme labels; U.S. Pat. No. 3,996,345 discloses fluorescer-quencher    labels; U.S. Pat. No. 4,062,733 discloses radioactive label; U.S.    Pat. No. 4,067,959 discloses fluorescer or enzyme label; U.S. Pat.    No. 4,104,099 discloses chemiluminescent label; and U.S. Pat. No.    4,160,645 discloses non-enzymatic catalyst label, U.S. Pat. No.    3,966,879 discloses an electrophoretic technique employing an    antibody zone and U.S. Pat. No. 4,120,945 discloses a radioimmune    assay (RIA) where labeled analyte is initially bound to a solid    support through antibody. U.S. Pat. No. 4,233,402 discloses enzyme    pair labels; U.S. Pat. No. 4,720,450 discloses chemically induced    fluorescent labels; and U.S. Pat. No. 4,287,300 discloses enzyme    anionic charge labels.

Labels can also be metal-containing sols; i.e., metal or metal compoundssuch as metal oxides, metal hydroxides, metal salts, metals ormetal-containing compounds mixed with polymers or coated onto polymernuclei. These metal labels may include dry forms of any of theabove-named metal or metal compound sols, and preferably includecolloidal gold in dry form.

“Label response” refers to the signal strength that is generated by thelabel when it becomes attached to the reagents immobilized in the testor standard bands. With color generating labels reflective light unitsare a preferable way of measuring label response.

-   9 . A “complex” means (depending on the context) any multimolecular    complex formed by analyte and one or more ligands, or by labeled    ligand and immobilized ligand. In a sandwich-type immunoassay, e.g.,    the following complexes occur: analyte/labeled ligand duplex first    produced in the assay (first complex) and analyte/labeled ligand    triplex formed second in the assay (second complex).-   “Fluid communication” refers to structures which are in contact with    but not necessarily affixed to one another and which allow the    passage of a fluid from one to the other.-   11 . “Assay” refers to several different types of assay formats in    which an analyte of interest can be detected using an assay analyte    test strip. For example, in a sandwich-type immunoassay, analytes of    interest in the analyte sample, when present, bind a labeled tracer    movably incorporated in the analyte test strip (consisting of a    porous membrane) at the tracer zone to form a first complex. The    tracer is a molecule, which binds the analyte of interest and is    conjugated to a label, preferably a metal label, and most preferably    colloidal old.-   12 . The term “test band”or “test zone” as used herein refers to a    region or zone on the chromatographic assay strip that contains at    least one analyte binding agent. The analyte binding agent is    usually immobilized in a band or zone such that after reaction with    a detectable agent, the band or zone produces an observable or    measurable result reflecting the presence or amount of analyte    present in the sample. The “test band” may be comprised of more than    one capture zone for capturing more than one analyte in the sample,    in which event, more than one analyte binding agent may be used. For    example, two assay combinations that are considered to be within the    scope of the invention are assay combinations that simultaneously    detect hepatitis C virus. (HCV) and human immunodeficiency virus    (HIV), and assay combinations that simultaneously detect Hepatitis B    surface antigen (HBsAg) and Treponema pallidum antigen (TP). Still    other combinations are possible and are within the scope of the    invention.-   13 . The term “conjugate” and “detectable agent” are used    interchangeably herein to refer to an antibody or an antigen that is    conjugated to a detectable material such as a colored agent, a    fluorescent agent, an enzyme agent or a chemiluminescent agent. In    the practice of the present invention, the “conjugate” or    “detectable agent” specifically binds the analyte to be determined    or the captured analyte immobilized on the capture band. Optionally,    the “conjugate” or “detectable agent” produces a measurable    quantitative reading at the capture band that reflects the amount of    an analyte present at the capture band. As described further below,    the direct measurable quantitative density in the capture band does    not necessarily reflect the amount of an analyte present at the    capture band through binding, but the intensity of band as measured    by reflective light units (RLU) does reflect the amount of an    analyte present at the capture band.-   14 . The term “standard band” or “calibrator band” as used herein    contains calibrator agents immobilized in calibrator binding zones    on the test strip. The calibrator agents bind specifically to    calibrator binding agents to form a calibrator binding pair. The    present invention includes two or more calibrator bands. An    advantage to having calibrator binding pairs is that they act as    internal standards, that is, the calibrator against which the amount    of an analyte present at the capture band may be calculated. The    calibrators may be used to correct for strip to strip variability.    One of the calibrators can be designated a high calibrator (“HC”)    and the other of the calibrators can be designated a low calibrator    (“LC”). The reflection density of HC and of LC, can be used to    determine the standard curve. It is also within the scope of this    invention to use more than standard bands all of different    concentrations of he xcalibrator agent. The standard curve is made    for each quantitative assays by using the RLU values of the    calibrator agents to generate a regression equation to describe the    relationship between two variables. Although, in general, any    conventional calibrators can be used herein, it is generally    preferred to use as calibrators compounds that do not exist in the    sample or do not immunologically cross-react with compounds that    exist in the sample; for example, monoclonal antibody against rabbit    immunoglobulin, monoclonal antibody against bovine thyroid globulin,    or antibody against 2,4-dinitrophenylated bovine serum albumin    (BSA-DNP), which can be purchased from Molecular Probes (Eugene,    Oreg., cat#A-23018) can be used as the two points of calibrator    reagent. The compound 2,4-dinitrophenol (DNP) is a small molecule    which does not exist within the human body but acts as a hapten;    that is, it is immunogenic when conjugated to a larger molecule such    as a protein carrier and injected into an antibody-producing mammal    such as a mouse, a rat, a cow, a rabbit, a horse, a sheep, or a    goat.

The test devices of the present invention comprise test strips, whichmay be enclosed in a cassette for easier use. Such test strips enclosedin such cassettes are described in U.S. Pat. Nos. 5,821,073 and5,384,264, the disclosures of which are hereby incorporated by thisreference. The test strips employed in the present invention generallycomprise a porous test membrane of sufficient porosity to allow thecomponents of the test including the analyte and its complexes to flowthrough the membrane by capillary action. Preferred membranes are porousnitrocellulose, porous, polypropylene and paper membranes. Suchmembranes are well known in the art. These membranes contain immobilizedligands capable of binding the analyte in a test zone, preferably in theform of a band across the width of the test membrane as well as the HCand LC calibrator agents in separate zones across the width of the testmembrane in the calibrator are standard bands. As indicated theimmobilized ligands used for the test and calibrator zones aredifferent. Known amounts of calibrator agents are immobilized in thestandard bands to establish the base line for the quantitativedetermination of the analyte. The remaining active sites after theapplication of the ligands in the various zones on the membrane areblocked to allow free flow of the analyte, analyte complexes, labeledligands and labeled ligand complexes through the test strip.

In addition to the porous membrane the test strips of the presentinvention contain a second porous membrane upstream from the firstmembrane but in fluid communication with the test membrane containingthe immobilized ligand bands.

The upstream membrane is of an inert type in that it does not containsites, which would bind ligands flowing through the membrane. Suchmembranes are preferably made of unwoven fiberglass or polypropylene,again of sufficient porosity to allow capillary flow of analyte, analytecomplexes and labeled ligands. These membranes can also be suitably usedto separate the analyte to be tested from components in the sample,which could interfere in the analysis of the analyte. Thus in theanalysis of blood the red and white blood cells can be separated fromthe fluid serum containing the analyte to be tested. The upstreammembrane therefore contains a site for the application of the sample,and in addition contains the labeled ligands, downstream from theapplication site and upstream from the contact with the test membrane,that react with the analyte in a sandwich assay or with the bound ligandin the test zone in a competitive assay, as well labeled ligands thatcomplex with the bound calibrator ligands in the standard bands. Thelabeled reagents are deposited on the upstream membrane in a manner thatthey can lift of from the membrane when contacted by the fluid sampleand in the case of the analyte react with the analyte to form a complexthat can then continue to flow from the upstream membrane into the testmembrane.

The test strip further contains a third membrane, in fluid connectionwith the test membrane, at the downstream end of the test membranecapable of absorbing the fluid sample that has passed through the testmembrane and that acts as the driving force to cause the fluid sample toflow through the test membrane by capillary action. Such adsorption padsare preferably made of high absorbency materials such as paper that arecapable of absorbing the sample and any buffer added to the strip.

The operation of the assay of the present invention basically involvesthe addition of a known quantity of a fluid sample containing asuspected analyte to the device at the sample application site using atransfer pipette. Generally the amount of sample will be in the range of10 to 100 microliters. Usually when the test strip is incorporated intoa cassette, a sample well is provided for the addition of the sample tothe test strip. The sample is absorbed by the upstream membrane anddrawn by capillary action through such upstream membrane towards thetest membrane. In the process the sample passes through the region ofthe upstream membrane where label bound ligand a are removablydeposited. In addition to the label ligand that binds to the analyte ifpresent the upstream membrane also contains high and low calibratorlabel ligands removably deposited on the upstream membrane. In apreferred embodiment the two calibrator label ligands and the labelligands used to bind to the analyte to be tested the sample are mixedand in dry form.

In the case of the sample being blood the upstream membrane can also actas a filter to filter out the red and white blood cells but let theserum or plasma pass through. The flow of the sample through the teststrip is frequently facilitated by the addition of a buffer. In generalthe amount of buffer added is at least equal to but no more than fivetimes the quantity of the fluid sample. Suitable buffers include anypharmaceutically acceptable aqueous buffer, which will not react withthe test sample of the other test and control ligands of the test strip.In general phosphate buffers, based on mono and disodiumhydrophosphateand commercially available, are preferred although other buffers such ascitrate buffers or Ringer's solution may also be used.

The sample stream picks up the label ligands for both the test andstandard bands before contacting the test membrane. The preferred labelsof the present invention are those that give rise to color complexes inthe test and standard bands. Although color resulting from enzyme boundligands or latex bound ligands which have been used in conjunction withqualitative assays using capillary flow of test strips such as disclosedin the above mentioned patents and others can be used in the assays ofthe present invention, the preferred color forming ligands are goldconjugates that can bind to the desired analyte to be analyzed and tothe calibrator agents. The quantitative assay of the present inventionis based on the color intensity of an analyte sample and the colorintensity generated by the calibrator agents in the standard bands. Thusin sandwich assay, the higher the amount of analyte in the test sample,the more label ligands binds to the analyte and the higher the amount oflabel ligand/analyte complex that will bind to the to the immobilizedligand in the test zone, increasing the label intensity in the band.Thus label intensity increases with increasing amount of analyte presentin the sample. In a competitive assay, the higher the amount of analytein the test sample, the less label ligand can bind to the immobilizedligand-BSA (analyte-BSA) in the test zone, decreasing the labelintensity in the test zone. Thus label intensity decreases withincreasing amount of analyte present in the sample. However in order toaccurately establish the actual concentration of the analyte, factorsother than analyte concentration must be excluded from any quantitativedetermination. It is for that purpose that the test strips of thepresent invention employ the calibrator agents in the standard bandswhich then provide the means for an accurate quantitative determination.Preferably and for higher accuracy the present invention employs atleast two different calibrator agents in separate standard zones, beforeand after the test zone on the test membrane. Since a fixed amount ofthe calibrator agent is immobilized in the standard zones and an excessof the calibrator label conjugate is deposited in the upstream band, thesame intensity of color is generated in the standard bands at any giventime interval time during the test for different test strips made in thesame way. Using the relative light units a standard curve can beobtained for each high and low calibrator zone, which then serves as thebase for the determination of the quantitative amount of analytepresent. Although, in general, any conventional calibrators can be usedherein, it is generally preferred to use as calibrators agents that donot exist in the sample or do not immunologically cross-react withcompounds that exist in the sample; for example, 2,4-dinitrophenylatedbovine serum albumin (BSA-DNP), which can be purchased from MolecularProbes (Eugene, Oreg., cat#A-23018) can be U.S.ed as the calibratorreagent. The compound 2,4-dinitrophenol (DNP) is a small molecule whichdoes not exist within the human body but acts as a hapten; that is, itis immunogenic when conjugated to a larger molecule such as a proteincarrier and injected into an antibody-producing mammal such as a mouse,a rat, a cow, a rabbit, a horse, a sheep, or a goat. Immobilized ligandsin the low calibrator standard zone are for example monoclonal antibodyagainst bovine thyroid globulin (BTG) and immobillied ligand a in thehigh calibrator standard zone are for example goat anti-rabbit IgGproteins. Conjugates used with the immobilized ligands are goat BTG goldantigens and rabbit IgG gold antigens.

In order to determine the amount of the analyte present it is necessaryto develop the relationship of color intensity in the test zone withconcentration of analyte in a sample. Such relationship, plotted as acurve, is obtained by preparing a solution or dispersion of the analyteto be tested at a known high concentration and then continuouslydiluting the test solution or dispersion and measuring the change incolor intensity in exposed test strips. Obviously this curve will alsobe different at different time intervals. However these curves can becorrelated with the standard curves obtained from the calibrator agentsfor known concentrations in the case of measurement of a samplecontaining an unknown amount of analyte. Thus in each test of an unknownsample three different color intensities are obtained. The intensitiesof the standard band can be correlated to the standard concentrationcurves for the analyte and a quantitative value for the analyte in thesample to be tested calculated. For verification purposes it isdesirable to obtain relative light units for two exposure times, whichcan be correlated to the standard values of concentration vs. labelresponse in reflectance units to obtain a verification of theconcentration of the analyte in the sample. The reliability of the curvedepends in large part on the accuracy and strip to strip consistency ofthe binding agent in the standard bands. Such accuracy and consistencyis accomplished by using linear motion displacement pumps that providedigitally controlled pulse free linear flows as are available from theIVEK corporation.

Although the necessary calibration of the standard strips andconcentration calculations can be carried out manually, they arepreferably embodied in software that can be read by commercial equipmentcapable of measuring the color intensity of the test band and thestandard bands. The color intensity data points generated by thestandard bands and known concentrations of analyte in the test band canbe incorporated into a memory device such as an electro-magnetic card oran RFID card. When such card is inserted into a commercially availableoptical camera reader, such as manufactured by Kaiwood Technology Co.together with a cassette that has been treated with a sample, the readercan provide the concentration of any analyte in the sample.

In general analytes and the corresponding complex forming antibodies, orantigen if the analyte is an antibody, are known in the art from thedevelopment of qualitative assays for such analytes, as are the labelconjugates that will bind to the analyte. For example, if the lateralflow assay i intended to measure hepatitis B virus (“HBV”) surfaceantigen (HBsAg) in a blood sample, the capture band will containantibody to HBsAg immobilized on the test membrane at the test band.

Suitable analytes include, but are not limited to antigens, antibodies,hormones, drugs, cell proteins, DNAs, cardiac markers, tumor or cancermarkers, autoimmune disease markers, or arty macromolecule that couldraise antibodies. When the analyte is an antigen, the antigen can be anantigen associated with an infectious agent. The infectious agent can bea virus, a bacterium, a fungus, or a prion. When the infectious agent isa virus, the virus can be selected from the group consisting of HIV,hepatitis virus A, B, C, and D, herpes simplex virus, cytomegalovirus,papilloma virus, Ebola vir, SARS virus Rhinovirus, and Vaccinia virus,but is not limited to those viruses. When the infectious agent is abacterium, the bacterium can be a gram-positive bacterium or agram-negative bacterium. The bacterium can be selected from the groupconsisting of Bacillus anthracis, Escherichia coli, Helicobacter pylori,Neisseria gonorrhoeae, Salmonella species, and Shigella species, but isnot limited to those bacteria. When the infectious agent is a fungus,the fungus can be a Mycosporum species or an Aspergillus species, but isnot limited to those fungi.

When the analyte is a hormone, typically it is selected from the groupconsisting of hCG, thyroxin, TSH, glucagons, insulin, relaxin,prolactin, luteinizing hormone, melanotropin, somatotropin,follicle-stimulating hormone, gastrin, bradykinin, vasopressin, andother releasing factors; however, other hormones of physiological orpathological interest can be the analyte.

When the analyte is a cancer or tumor marker, typically it is selectedfrom the group consisting of prostate specific antigen (PSA),carcinoembryonic antigen (CEA), and α-fetoprotein; however, other canceror tumor markers can be the analyte.

When the analyte is a cardiac marker, the cardiac marker is typicallyselected from the group consisting of Troponin-I, Troponin T, Creatinekinase-MB isoforms (CK-MB), myoglobin, C-reactive protein (CRP), fattyacid binding protein (FABP), glycogen phosphorylase isoenzyme BB (GPBB),B-type natriuretic peptide (BNP) and pro-BNP; however, the analyte canbe another cardiac marker.

The invention is further illustrated but not limited by the followingexamples.

EXAMPLE 1

Onto a test strip comprising a porous nitrocellulose strip (7.5 cm×0.7cm) as the test membrane sufficient monoclonal antibody against humanprostrate specific antigen (PSA) is immobilized in a test band to giverise to measurable color intensity when complexed with PSA analyte colorconjugates that can be expected in a sample. In separate experiments therelationship of color intensity and concentration is established as acurve. Rabbit IgG and bovine thyroid globulin are immobilized in twostandard bands by using an IVEK ceramic linear pump having an accuracyof 99.95%, in known amounts sufficient to give rise to a color intensitycorresponding to PSA at concentration of 1 and 10 nanogram permilliliter (ng/ml) respectively when such is complexed with a gold labelmonoclonal PSA specific antibody. The test membrane is dried in adehumidifying chamber (humidity of 10-15%) overnight, Excess of the goldlabel antibody against PSA, gold label monoclonal antibody againstrabbit IgG and gold label monoclonal antibody against bovine thyroidglobulin are also removable deposited in an upstream fiberglassmembrane. The conjugate fiberglass pad is treated with a buffer toneutralize interfering sites and then vacuum dried at 2 Torr overnightat room temperature. The pad was then cut into narrow strips (1×0.7 cm).The bands are arranged such that the sample first contacts the lowcalibrator band showing the color intensity with the gold conjugatecorresponding to 1 ng/ml of PSA antigen, then the test band, followed bythe high calibrator agent band, capable of binding the equivalent of 10ng/ml of PSA. The correlation of the color intensity in RLU units withanalyte concentration, lot #, test time, slope and intercept for the twostandard bands of the particular test strip is loaded into a RFID memorycard. The antigen concentrations can then be calculated using theequation Y=AX+B where Y is the concentration of the analyte, X is thecolor intensity generated by the sample in RLU units, A is the slope ofthe curve obtained from the standard values and B is the intercept ofthe test value on the curve. The test membrane is in fluid connectionwith an upstream non-woven fiberglass membrane onto which is depositedthe antibody to the PSA antigen conjugated to a gold label by techniquesknown in the art. The label conjugate is deposited down stream fromwhere the sample to be analyzed is added to the upstream fiberglassmembrane. The assay test strip also Contains a sample absorption pad influid connection with the downstream end of the test membrane. The teststrip is inserted into a cassette such as described in U.S. Pat. No.5,821,073.

One drop of blood suspected of containing the PSA analyte, approximately30 microliters, is added to the sample applicator site on the upstreamfiberglass membrane followed by four drops, about 200 microliters, of acommercially available phosphate buffer. The fiberglass membrane issufficiently dense to filter out the red and white blood cells but willlet the serum pass. The serum of the blood combined with buffer flowsthrough the upstream membrane by capillary action to the label conjugateredistributing the gold antibody conjugate in the serum which then formsa complex with any analyte present in the sample. The buffer dilutedserum passes from the fiberglass membrane to the porous nitrocellulosemembrane and continues to flow by capillary action through thenitrocellulose membrane. Analyte gold conjugate complex is captured bythe PSA antibody deposited in the test band and gives rise to a reddishcolor the intensity of which corresponds to the amount of analytepresent in the sample. Monoclonal antibody against rabbit IgG andmonoclonal antibody against bovine thyroid globulin gold conjugateremovably deposited on the fiberglass membrane and contained in theserum are also captured by the high and low standard bands containingsufficient calibrator agent to capture the equivalent of either one orten ng/ml of analyte. In the presence of PSA analyte in the sample threebands of differing color intensity are obtained. After passing the testand standard bands the remaining buffered sample passes from thenitrocellulose strip to the absorption pad and is stored there. Thecassette with the color bands is then inserted into a reader togetherwith the memory card containing the correlation between color intensityas measured by relative light units (RLU) and concentration of analyte.The photo-camera reader employed, Kaiwood CHR 100 , is capable ofconverting the color intensity into concentration using the data on thememory card. Color intensities were measured 10 and 15 minutes after theaddition of the sample and buffer. The following results are obtained:

10 minutest test result: Analytes PSA Concentration RLU Calibrator H 10ng/ml 10 Calibrator L  1 ng/ml 1 Sample Y 4.04 Based on the two pointstandard curve, the reader showed 4 ng/ml of the PSA antigen (Y) in thesample.

15 minutes test result: Analytes PSA Concentration RLU Calibrator H 10ng/ml 23.9 Calibrator L  1 ng/ml 10.3 Sample Y 19.49 Based on the twopoint standard curve, the reader showed 4 ng/ml of PSA antigen (Y) inthe sample.

EXAMPLE 2

In a procedure similar to that of Example 1, onto a test stripcomprising a porous nitrocellulose strip (7.5 cm×0.7 cm) as the testmembrane containing sufficient monoclonal antibody against human thyroidstimulate hormone antigen (TSH) is immobilized in the test band to giverise to a measurable color intensity at TSH concentrations that can beexpected in the sample. In separate prior experiments the relationshipof TSH concentration and color intensity is established. Rabbit IgG andbovine thyroid globulin are immobilized in two standard bands by usingan IVEK dispensing linear pump, in amounts sufficient to give rise to ameasurable color intensity corresponding to TSH at concentrations of 5μIU/ml and 20 μIU/ml respectively when complexed with a gold labelmonoclonal TSH. The test membrane is dried in dehumidifying chamber(humidity:10-15%) overnight. Excess of the gold label antibody againstTSH, gold label monoclonal antibody against rabbit IgG and gold labelmonoclonal antibody against bovine thyroid globulin are also removablydeposited in an upstream fiberglass membrane. The conjugate fiberglassmembrane was then treated with a conjugate buffer to neutralize anyinterfering sites and then vacuum dried at 2 Torr overnight at roomtemperature. The pad was then cut into (1×0.7 cm) strips. The bands arearranged such that the sample first contacts the low calibrator bandshowing the color intensity with the gold conjugate corresponding to 5μIU/ml of TSH antigen, then the test band, followed by the highcalibrator agent band, capable of binding the equivalent of 20 μIU/ml ofTSH. The correlation of the color density in RLU units with analyteconcentration, lot #, test time, slope and intercept for the twostandard bands of the particular test strip is loaded into a RFID memorycard. The antigen concentrations can then be calculated using theequation Y=AX+B where Y is the concentration of the analyte, X the colorintensity generated by the sample in RLU units, A is the slope of thecurve obtained from the standard values and B is the intercept of thetest value on the curve.

The test membrane is in fluid connection with an upstream non-wovenfiberglass membrane onto which is deposited the antibody to the TSHantigen conjugated to a gold label by techniques known in the art. Thelabel conjugate is deposited down stream from where the sample to beanalyzed is added to the upstream fiberglass membrane. The assay teststrip also contains a sample absorption pad in fluid connection with thedownstream end of the test membrane. The test strip is inserted into acassette such as described in U.S. Pat. No. 5,821,073.

One drop of blood suspected of containing the TSH analyte, approximately30 microliters, is added to the sample applicator site on the upstreamfiberglass membrane followed by four drops, about 200 microliters, of acommercially available phosphate buffer. The fiberglass membrane issufficiently dense to filter out the red and white blood cells but willlet the serum pass. The serum of the blood combined with buffer flowsthrough the upstream membrane by capillary action to the label conjugateredistributing the gold antibody conjugate in the serum which then formsa complex with any analyte present in the sample. The buffer dilutedserum passes from the fiberglass membrane to the porous nitrocellulosemembrane and continues to flow by capillary action through thenitrocellulose membrane. Analyte gold conjugate complex is captured bythe TSH antibody deposited in the test band and gives rise to a reddishcolor the intensity of which corresponds to the amount of analytepresent in the sample. Monoclonal antibody against rabbit IgG andmonoclonal antibody against bovine thyroid globulin gold conjugateremovably deposited on the fiberglass membrane and distributed in theserum are also captured by the high and low standard bands containingsufficient calibrator agent to capture the equivalent of either 5 or 205 μIU/ml of analyte. In the presence of TSH analyte in the sample threebands of differing color intensity are obtained. After passing the testand standard bands the remaining buffered sample passes from thenitrocellulose strip to the absorption pad and is stored there. Thecassette with the color bands is then inserted into a reader togetherwith the memory card containing the correlation between color intensityas measured by relative light units (RLU) and concentration of analyte.The photo-camera reader employed, Kaiwood CHR 100, is capable ofconverting the color intensity into concentration using the data on thememory card. Color intensities were measured 15 minutes after theaddition of the sample and buffer. The following results are obtained:

15 minutest test result: Analytes TSH Concentration RLU Calibrator H 20μIU/ml 102 Calibrator L  5 μIU/ml 47 Sample Y 19.5 Based on the twopoint standard curve, the reader showed 0.78 μIU/ml of the TSH antigen(Y) in the sample.

EXAMPLE 3

The procedure of Example 1 is repeated to determine the concentration ofhuman carcinoembryonic antigen (CEA) with gold label monoclonal specificanti bodies. In separate prior experiments the relationship of CEAconcentration to color intensity generated by the antibody when combinedwith the analyte is established. Rabbit IgG and bovine thyroid globulinare immobilized in two standard bands by using an IVEK ceramic linearpump having an accuracy of 99.95%, in amounts sufficient to give rise toa color intensity when complexed with old label antibodies correspondingto concentration of 4 and 100 ng/ml respectively CEA when such iscomplexed with a gold label monoclonal CEA specific antibody, an excessof which is removably deposited on the upstream fiberglass membrane. CEAspecific antibody is immobilized in the test band in sufficientconcentration to provide measurable color intensities when complexedwith labeled CEA concentrations expected to be present in the sample.Excess of the gold label antibody against CEA gold label monoclonalantibody against rabbit IgG and gold label monoclonal antibody againstbovine thyroid globulin are also removably deposited in an upstreamfiberglass membrane. As in Example 1 the correlation of colorintensities to antigen concentration is established using the equationY=AX+B.

One drop of blood suspected of containing the CEA analyte, approximately30 microliters, is added to the sample applicator site on the upstreamfiberglass membrane followed by 4 drops, about 200 microliters, of atesting phosphate buffer. The fiberglass membrane used is sufficientlydense to filter out the red and white blood cells but will let the serumpass. The serum of the blood combined with buffer flows through theupstream membrane by capillary action to the label conjugateredistributing the gold antibody conjugate which then forms a complexwith any analyte present in the sample. The buffer diluted serum passesfrom the fiberglass membrane to the porous nitrocellulose membrane andcontinues to flow by capillary action through the nitrocellulosemembrane. Analyte gold conjugate complex is captured by the CEA antibodydeposited in the test band and gives rise to a reddish color theintensity of which corresponds to the amount of analyte present in thesample. Monoclonal antibody against rabbit IgG and monoclonal antibodyagainst Bovine thyroid globulin gold conjugate removably deposited onthe fiberglass membrane and picked up by the serum are also captured bythe high and low standard bands. In the presence of CEA analyte in thesample three bands of differing color intensity are obtained. Afterpassing the test and standard bands the remaining buffered sample passesfrom the nitrocellulose strip to the absorption pad and is stored there.The cassette with the color bands is then inserted into a RFID readertogether with the memory card containing the correlation between colorintensity as measured by relative light units (RLU) and concentration ofanalyte. The reader employed, CER 100, is capable of converting thecolor intensity into concentration using the data on the memory card.Color intensities were measured at 15 minutes after the addition of thesample and buffer. The following results are obtained:

15 minutest test result: Analytes CEA Concentration RLU Calibrator H 100ng/ml 78 Calibrator L  4 ng/ml 21.4 Sample Y 34.2 Based on the two pointstandard curve, the reader showed 25 ng/ml of the CEA antigen (Y) in thesample.

EXAMPLE 4

The procedure of Example 1 is repeated to determine the concentration ofhuman Troponin-I antigen (TNI-I) in a sample using gold label monoclonalspecific TNI-I antibody. The relationship of TNI-I concentration and thecolor intensity generated by the antibody when conjugated to the analyteis established in prior experiments. Rabbit IgG and bovine thyroidglobulin are immobilized in two standard bands by using an IVEK ceramiclinear pump having an accuracy of 99.95%, in amounts sufficient to giverise to a measurable color intensity corresponding to TNI-Iconcentrations of 1 and 50 ng/ml respectively when such is complexedwith a gold label monoclonal TNI-I specific antibody. TNI-I specificantibody is immobilized in the test band. Excess of the gold labelantibody against TNI-I, gold label monoclonal antibody against rabbitIgG and gold label monoclonal antibody against bovine thyroid globulinare also removably deposited in an upstream fiberglass membrane. As inExample 1 the correlation of color intensity to antigen concentration isestablished using the equation Y=AX+B.

One drop of blood suspected of containing the TNI-I analyte,approximately 30 microliters, is added to the sample applicator site onthe upstream fiberglass membrane followed by 4 drops, about 200microliters, of a testing phosphate buffer. The fiberglass membrane usedis sufficiently dense to filter out the red and white blood cells butwill let the serum pass. The serum of the blood combined with bufferflows through the upstream membrane by capillary action to the labelconjugates redistributing the gold antibody conjugates which can form acomplexes with any analyte present in the sample and with the antigensin the standard band. The buffer diluted serum passes from thefiberglass membrane to the porous nitrocellulose membrane and continuesto flow by capillary action through the nitrocellulose membrane. Analytegold conjugate complex is captured by the TNI-I antibody deposited inthe test band and gives rise to a reddish color the intensity of whichcorresponds to the amount of analyte present in the sample. Monoclonalantibodies against rabbit IgG and against bovine thyroid globulin goldconjugates removably deposited on the fiberglass membrane and picked upby the serum are captured by the high and low standard bands. Afterpassing the test and standard bands the remaining buffered sample passesfrom the nitrocellulose strip to the absorption pad and is stored there.The cassette with the color bands is then inserted into a readertogether with the memory card containing the correlation between colorintensity as measured by relative light units (RLU) and concentration ofanalyte. The reader employed, CHR 100 is capable of converting the colorintensity into concentration using the data on the memory card. Colorintensities were measured at 15 minutes after the addition of the sampleand buffer. The following results are obtained:

15 minutest test results: Analytes TNI-I Concentration RLU Calibrator H50 ng/ml 62 Calibrator L  1 ng/ml 12 Sample Y 6 Based on the two pointstandard curve, the reader showed 0.5 ng/ml of the CEA antigen (Y) inthe sample.

EXAMPLE 5

The procedure of example 5 is competitive assay procedure to determinethe concentration of human Thyroxin antigen (T-4) in a blood or serumsample. In separate experiments the relationship of T-4 concentration tocolor intensity of the gold labeled conjugate is established. In aprocedure similar to that of Example 1 rabbit IgG and bovine thyroidglobulin are immobilized in two standard bands by using an IVEK ceramiclinear pump having an accuracy of 99.95%, in amounts sufficient to giverise to a measurable color intensity corresponding to T-4 concentrationsof 5 and 20 ng/ml respectively is when such is complexed with a goldlabel monoclonal (T-4) specific antibody and complexed to the antibodyin the test band. Furthermore T-4 specific monoclonal antibodies arealso deposited in the test band of the nitrocellulose strip. The bandsare arranged such that the sample first contacts the low calibrator bandshowing the color intensity with the gold conjugate corresponding to 5μg/ml of T-4 antigen, then the test band, followed by the highcalibrator agent band, capable of binding the equivalent of 20 μg/ml ofT-4 . The deposited antigen (T-4) conjugate with bovine serum albumin inthe test band are allowed to react with the gold sol conjugate ofT4-antibody on the nitrocellulose and become permanently immobilized intest bands on the test membrane after reaction. The assay strip is thenexposed to the gold conjugate monoclonal antibody against rabbit IgG andmonoclonal antibody against bovine thyroglobulin to generate thestandard color intensities from which a standard curved can begenerated. The standard curve showing the correlation of color intensitywith analyte concentration for different lots of assay strips is loadedinto a RFID memory card. The test value may be calculated by using theequation of Y=AX+B as set forth in Example 1. The test membrane is influid connection with an upstream non-woven fiberglass membrane ontowhich is deposited the antibody to the T-4 antigen conjugated to a goldlabel by techniques known in the art. The label conjugate is depositeddown stream from where the sample to be analyzed is added to theupstream fiberglass membrane. The assay test strip also contains asample absorption pad in fluid connection with the downstream end of thetest membrane. The test strip is inserted into a cassette such asdescribed in U.S. Pat. No. 5,821,073.

One drop of blood suspected of containing the T-4 analyte, approximately30 microliters, is added to the sample applicator site on the upstreamfiberglass membrane followed by 4 drops, about 200 microliters, of atesting phosphate buffer. The fiberglass membrane used is sufficientlydense to filter out the red and white blood cells but will let the serumpass. The serum of the blood combined with buffer flows through theupstream membrane by capillary action to the label conjugateredistributing the gold antibody conjugate which then forms a complexwith any analyte present in the sample. The buffer diluted serum passesfrom the fiberglass membrane to the porous nitrocellulose membrane andcontinues to flow by capillary action through the nitrocellulosemembrane. Analyte gold conjugate complex is captured by the T-4 antibodydeposited in the test band and gives rise to a reddish color theintensity of which corresponds to the amount of analyte present in thesample. The corresponding standard band gold conjugate antibodies,removably deposited on the fiberglass and picked up by the sample, arecaptured by the antigens in the standard bands. After passing the testand standard bands the remaining buffered sample passes from thenitrocellulose snip to the absorption pad and is stored there. Thecassette with the color bands is then inserted into a reader togetherwith the memory card containing the correlation between color intensityas measured by relative light units (RLU) and concentration of analyte.The reader employed, CHR 100 is capable of converting the colorintensity into concentration using the data on the memory card. Colorintensities were measured at 15 minutes after the addition of the sampleand buffer. The following results are obtained:

15 minutest test result: Analytes T-4 Concentration RLU Calibrator H 20μg/ml 39 Calibrator L  5 μg/ml 79 Sample Y 66 Based on the two pointstandard curve, the reader showed 10 μg/ml of the T-4 antigen (Y) in thesample.

EXAMPLE 6

Onto a test strip comprising a porous nitrocellulose strip (7.5 cm×0.7cm) as the test membrane sufficient monoclonal antibody against humanprostrate specific antigen

(PSA) in a test band to give rise to measurable color intensity whencomplexed with PSA analyte color conjugates that can be expected in asample. Rabbit IgG, bovine thyroid globulin and murine hybridoma areimmobilized in three standard bands by using an IVEK ceramic linear pumphaving an accuracy of 99.95%, in amounts sufficient to give rise to acolor intensity corresponding to a PSA concentration of 1, 5 and 10ng/ml respectively when such is complexed with a gold label monoclonalPSA specific antibody. The test membrane is dried in a dehumidifyingchamber (humidity of 10-15%) overnight, Excess of the gold labelantibody against PSA, gold label monoclonal antibody against rabbit IgG,anti-mouse antibody capable of complexing with hybridoma and gold labelmonoclonal antibody against bovine thyroid globulin are also removabledeposited in an upstream fiberglass membrane. The conjugate fiberglasspad is coated with the label conjugates, then treated with a buffer toneutralize interfering sites and then vacuum dried at 2 Torr overnightat room temperature. The pad is then cut into narrow strips (1×0.7 cm).The bands are arranged such that the sample first contacts the low andintermediate calibrator bands showing the color intensity with the goldconjugate corresponding to 1 and 5 ng/ml of PSA antigen, then the testband, followed by the high calibrator agent band, capable of binding theequivalent of 10 ng/ml of PSA. The correlation of the color density inRLU units with analyte concentration, lot #, test time, slope andintercept for the three standard bands of the particular test strip isloaded into a RFID memory card. The antigen concentrations can then becalculated using the equation Y=AX+B where Y is the concentration of theanalyte, X is the color intensity generated by the sample in RLU units,A is the slope of the curve obtained from the standard values and B isthe intercept of the test value on the curve.

The test membrane is in fluid connection with an upstream non-wovenfiberglass membrane onto which is deposited the antibody to the PSAantigen conjugated to a gold label by techniques known in the art. Thelabel conjugate is deposited down stream from where the sample to beanalyzed is added to the upstream fiberglass membrane. The assay teststrip also contains a sample absorption pad in fluid connection with thedownstream end of the test membrane. The test strip is inserted into acassette such as described in U.S. Pat. No. 5,821,073.

One drop of blood suspected of containing the PSA analyte, approximately30 microliters, is added to the sample applicator site on the upstreamfiberglass membrane followed by four drops, about 200 microliters, of acommercially available phosphate buffer. The fiberglass membrane issufficiently dense to filter out the red and white blood cells but willlet the serum pass. The serum of the blood combined with buffer flowsthrough the upstream membrane by capillary action to the label antibodyconjugate location where the analyte present in the sample complexeswith the gold antibody and where the buffer diluted sample picks up theantibodies of the antigens in the standard bands. The buffer dilutedserum passes hum the fiberglass membrane to the porous nitrocellulosemembrane and continues to flow by capillary action through thenitrocellulose membrane. Analyte gold conjugate complex is captured bythe PSA antibody deposited in the test band and gives rise to a reddishcolor the intensity of which corresponds to the amount of analytepresent in the sample. Monoclonal antibody against rabbit IgG, antimouseantibody and monoclonal antibody against bovine thyroid globulin goldconjugate deposited on the fiberglass membrane and picked up by thesample are also captured by the high, intermediate and low standardbands containing sufficient calibrator agent to capture the equivalentof either one, five or ten ng/ml of analyte. In the presence of PSAanalyte in the sample four bands of normally differing color intensityare obtained. After passing the test and standard bands the remainingbuffered sample passes from the nitrocellulose strip to the absorptionpad and is stored there. The cassette with the color bands is theninserted into a reader together with the memory card containing thecorrelation between color intensity as measured by relative light units(RLU) and concentration of analyte. The photo-camera reader employed,Kaiwood CHR 100, is capable of converting the color intensity intoconcentration using the data on the memory card. Color intensities weremeasured 10 and 15 minutes after the addition of the sample and buffer.Essentially the same results as obtained in Example 1 are obtained.

The invention claimed is:
 1. An assay device for the quantitativedetermination of an analyte, comprising a test strip containing a poroustest membrane allowing for capillary flow of the analyte and complexesof the analyte, a porous upstream membrane in fluid connection with thetest membrane and a porous downstream membrane in fluid connection withthe test membrane, (a) said test membrane comprising (i) a test bandsite having immobilized thereon a ligand capable of competing with theanalyte for binding a label conjugate capable of reacting with theanalyte to the test band site, and, (ii) a first standard band sitehaving immobilized thereon a first calibrator agent corresponding to afirst known high concentration of the analyte, and a second standardband site having immobilized thereon a second calibrator agentcorresponding to a second known low concentration of the analyte,wherein the two calibrator agents are different and are capable ofreacting with different label conjugates and binding such to thestandard band sites to provide a known label response in the standardband sites, (b) said upstream membrane having a site for the applicationof a sample to be analyzed, and having a site downstream from the sampleapplication site for depositing the label conjugate capable of reactingwith the analyte and depositing the two label conjugates capable ofreacting with the immobilized calibrator agents in the standard bands,and (c) said downstream membrane capable of absorbing said sample andproviding the capillary flow for the sample through the upstream andtest membrane.
 2. The device of claim 1, wherein the label conjugatesgive rise to a color.
 3. The device of claim 2, wherein the labelconjugates comprise gold conjugates, fluorescent dye conjugates, latexconjugates and enzyme conjugates.
 4. The device of claim 1, wherein thecalibrator agents are rabbit IgG and bovine thyroid globulin.
 5. Thedevice of claim 1, wherein the test strip is contained in a cassetteallowing for the addition of the sample at the upstream membrane and forviewing the results of color formation at the sites of the testmembrane.
 6. The device of claim 1, wherein the label conjugates for theanalyte and for the calibrator agents are different.
 7. The device ofclaim 1, wherein the test membrane is microporous nitrocellulose and theupstream membrane is fiberglass.
 8. The device of claim 1, wherein thetest membrane further comprises a third standard band.
 9. The device ofclaim 1, wherein the label conjugates generate visual signals at thetest band site and standard band sites.
 10. The device of claim 9,wherein the visual signals are generated by a colloidal gold conjugate,a fluorescent dye conjugate, or a latex particle.
 11. The method ofclaim 1, wherein the analyte is a cardiac marker.
 12. The device ofclaim 1, wherein the analyte is T4, the test band site is immobilizedwith T4-bovine serum albumin conjugate.